A segmentation clock operating in blastoderm and germband stages of Tribolium development.
ABSTRACT: In Drosophila, all segments form in the blastoderm where morphogen gradients spanning the entire anterior-posterior axis of the embryo provide positional information. However, in the beetle Tribolium castaneum and most other arthropods, a number of anterior segments form in the blastoderm, and the remaining segments form sequentially from a posterior growth zone during germband elongation. Recently, the cyclic nature of the pair-rule gene Tc-odd-skipped was demonstrated in the growth zone of Tribolium, indicating that a vertebrate-like segmentation clock is employed in the germband stage of its development. This suggests that two mechanisms might function in the same organism: a Drosophila-like mechanism in the blastoderm, and a vertebrate-like mechanism in the germband. Here, we show that segmentation at both blastoderm and germband stages of Tribolium is based on a segmentation clock. Specifically, we show that the Tribolium primary pair-rule gene, Tc-even-skipped (Tc-eve), is expressed in waves propagating from the posterior pole and progressively slowing until they freeze into stripes; such dynamics are a hallmark of clock-based segmentation. Phase shifts between Tc-eve transcripts and protein confirm that these waves are due to expression dynamics. Moreover, by tracking cells in live embryos and by analyzing mitotic profiles, we found that neither cell movement nor oriented cell division could explain the observed wave dynamics of Tc-eve. These results pose intriguing evolutionary questions, as Drosophila and Tribolium segment their blastoderms using the same genes but different mechanisms.
Project description:In Drosophila, a hierarchy of maternal, gap, pair-rule, and segment polarity gene interactions regulates virtually simultaneous blastoderm segmentation. For the last decade, studies have focused on revealing the extent to which Drosophila segmentation mechanisms are conserved in other arthropods where segments are added sequentially from anterior to posterior in a cellular environment. Despite our increased knowledge of individual segmentation genes, details of their interactions in non-Drosophilid insects are not well understood. We analyzed the Tribolium orthologs of Drosophila pair-rule genes, which display pair-rule expression patterns. Tribolium castaneum paired (Tc-prd) and sloppy-paired (Tc-slp) genes produced pair-rule phenotypes when their transcripts were severely reduced by RNA interference. In contrast, similar analysis of T. castaneum even-skipped (Tc-eve), runt (Tc-run), or odd-skipped (Tc-odd) genes produced severely truncated, almost completely asegmental phenotypes. Analysis of interactions between pair-rule components revealed that Tc-eve, Tc-run, and Tc-odd form a three-gene circuit to regulate one another as well as their downstream targets, Tc-prd and Tc-slp. The complement of primary pair-rule genes in Tribolium differs from Drosophila in that it includes Tc-odd but not Tc-hairy. This gene circuit defines segments sequentially in double segment periodicity. Furthermore, this single mechanism functions in the early blastoderm stage and subsequently during germ-band elongation. The periodicity of the Tribolium pair-rule gene interactions reveals components of the genetic hierarchy that are regulated in a repetitive circuit or clock-like mechanism. This pair-rule gene circuit provides insight into short-germ segmentation in Tribolium that may be more generally applicable to segmentation in other arthropods.
Project description:Embryonic anterior-posterior patterning is well understood in Drosophila, which uses 'long germ' embryogenesis, in which all segments are patterned before cellularization. In contrast, most insects use 'short germ' embryogenesis, wherein only head and thorax are patterned in a syncytial environment while the remainder of the embryo is generated after cellularization. We use the wasp Nasonia (Nv) to address how the transition from short to long germ embryogenesis occurred. Maternal and gap gene expression in Nasonia suggest long germ embryogenesis. However, the Nasonia pair-rule genes even-skipped, odd-skipped, runt and hairy are all expressed as early blastoderm pair-rule stripes and late-forming posterior stripes. Knockdown of Nv eve, odd or h causes loss of alternate segments at the anterior and complete loss of abdominal segments. We propose that Nasonia uses a mixed mode of segmentation wherein pair-rule genes pattern the embryo in a manner resembling Drosophila at the anterior and ancestral Tribolium at the posterior. DOI: http://dx.doi.org/10.7554/eLife.01440.001.
Project description:Thehead gap gene buttonhead (btd) is required for the patterning of head segments in the early Drosophila embryo. Mutant phenotypes of btd display a gap-like phenotype in which antennal, intercalary, mandibular and the anterior portion of the maxillary segmentsare eliminated. In agreement with the phenotypes, btd is expressed in a stripe covering the head segments at the blastoderm stage. During the early phase of the germband extension, btd is expressed in stripes with single segmental periodicity, which is required for the formation of the peripheral nervous system. In contrast to the key role of btd in Drosophila embryonic development, it has been suggested that Tribolium ortholog of btd (Tc-btd) is dispensable for embryonic head development. In order for better understanding of the requirement of Tc-btd in the early Tribolium embryo, we re-analyzed the expression patterns and functions of Tc-btd during embryonic segmentation. Tc-btd is expressed in segmental stripes at the stages of blastoderm and germband elongation. Up to 28.3% of embryos in which Tc-btd is knocked down displays the loss of antennal, mandibular and the pregnathal regions in the head, with abdominal segments being disrupted in the trunk. Our findings suggest that Tc-btd is required for the head and trunk development in the early Tribolium embryo.
Project description:In the short-germ beetle Tribolium castaneum, waves of pair-rule gene expression propagate from the posterior end of the embryo towards the anterior and eventually freeze into stable stripes, partitioning the anterior-posterior axis into segments. Similar waves in vertebrates are assumed to arise due to the modulation of a molecular clock by a posterior-to-anterior frequency gradient. However, neither a molecular candidate nor a functional role has been identified to date for such a frequency gradient, either in vertebrates or elsewhere. Here we provide evidence that the posterior gradient of Tc-caudal expression regulates the oscillation frequency of pair-rule gene expression in Tribolium. We show this by analyzing the spatiotemporal dynamics of Tc-even-skipped expression in strong and mild knockdown of Tc-caudal, and by correlating the extension, level and slope of the Tc-caudal expression gradient to the spatiotemporal dynamics of Tc-even-skipped expression in wild type as well as in different RNAi knockdowns of Tc-caudal regulators. Further, we show that besides its absolute importance for stripe generation in the static phase of the Tribolium blastoderm, a frequency gradient might serve as a buffer against noise during axis elongation phase in Tribolium as well as vertebrates. Our results highlight the role of frequency gradients in pattern formation.
Project description:Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). We use data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, our simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. Our results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects.
Project description:Long-germ insects, such as the fruit fly Drosophila melanogaster, pattern their segments simultaneously, whereas short-germ insects, such as the beetle Tribolium castaneum, pattern their segments sequentially, from anterior to posterior. While the two modes of segmentation at first appear quite distinct, much of this difference might simply reflect developmental heterochrony. We now show here that, in both Drosophila and Tribolium, segment patterning occurs within a common framework of sequential Caudal, Dichaete, and Odd-paired expression. In Drosophila these transcription factors are expressed like simple timers within the blastoderm, while in Tribolium they form wavefronts that sweep from anterior to posterior across the germband. In Drosophila, all three are known to regulate pair-rule gene expression and influence the temporal progression of segmentation. We propose that these regulatory roles are conserved in short-germ embryos, and that therefore the changing expression profiles of these genes across insects provide a mechanistic explanation for observed differences in the timing of segmentation. In support of this hypothesis we demonstrate that Odd-paired is essential for segmentation in Tribolium, contrary to previous reports.
Project description:Drosophila undergoes a form of development termed long germ segmentation, where all segments are specified nearly simultaneously so that by the blastoderm stage, the entire body plan has been determined. This mode of segmentation is evolutionarily derived. Most insects undergo short or intermediate germ segmentation, where only anterior segments are specified early, and posterior segments are sequentially specified during germband elongation. These embryological differences imply that anterior and posterior segments might rely upon different molecular mechanisms. In Drosophila, embryos mutant for giant show a gap in the anterior as well fusions of several abdominal segments. In Tribolium, a short germ beetle, giant is required for segmental identity, but not formation, in gnathal segments and also for segmentation of the entire abdomen. This raises the possibility that giant might not act as a gap gene in short and intermediate germ insects. Oncopeltus fasciatus is an intermediate germ insect that is an outgroup to the clade containing Drosophila and Tribolium. We cloned the Oncopeltus homolog of giant and determined its expression and function during segmentation. We find that Oncopeltus giant is a canonical gap gene in the maxillary and labial segments and also plays a gap-like role in the first four abdominal segments. Our results suggest that giant was a bona fide gap gene in the ancestor of these insects with this role being lost in the lineage leading towards Tribolium. This highlights the conservation of anterior patterning and evolutionary plasticity of the genetic regulation controlling posterior segmentation, even in short and intermediate germ insects.
Project description:<h4>Background</h4>The Drosophila larval head is evolutionarily derived at the genetic and morphological level. In the beetle Tribolium castaneum, development of the larval head more closely resembles the ancestral arthropod condition. Unlike in Drosophila, a knirps homologue (Tc-kni) is required for development of the antennae and mandibles. However, published Tc-kni data are restricted to cuticle phenotypes and Tc-even-skipped and Tc-wingless stainings in knockdown embryos. Hence, it has remained unclear whether the entire antennal and mandibular segments depend on Tc-kni function, and whether the intervening intercalary segment is formed completely. We address these questions with a detailed examination of Tc-kni function.<h4>Results</h4>By examining the expression of marker genes in RNAi embryos, we show that Tc-kni is required only for the formation of the posterior parts of the antennal and mandibular segments (i.e. the parasegmental boundaries). Moreover, we find that the role of Tc-kni is distinct in these segments: Tc-kni is required for the initiation of the antennal parasegment boundary, but only for the maintenance of the mandibular parasegmental boundary. Surprisingly, Tc-kni controls the timing of expression of the Hox gene Tc-labial in the intercalary segment, although this segment does form in the absence of Tc-kni function. Unexpectedly, we find that the pair-rule gene Tc-even-skipped helps set the posterior boundary of Tc-kni expression in the mandible. Using the mutant antennaless, a likely regulatory Null mutation at the Tc-kni locus, we provide evidence that our RNAi studies represent a Null situation.<h4>Conclusions</h4>Tc-kni is required for the initiation of the antennal and the maintenance of the mandibular parasegmental boundaries. Tc-kni is not required for specification of the anterior regions of these segments, nor the intervening intercalary segment, confirming that Tc-kni is not a canonical 'gap-gene'. Our finding that a gap gene orthologue is regulated by a pair rule gene adds to the view that the segmentation gene hierarchies differ between Tribolium and Drosophila upstream of the pair rule gene level. In Tribolium, as in Drosophila, head and trunk segmentation gene networks cooperate to pattern the mandibular segment, albeit involving Tc-kni as novel component.
Project description:wingless (wg)/Wnt family are essential to development in virtually all metazoans. In short-germ insects, including the red flour beetle (Tribolium castaneum), the segment-polarity function of wg is conserved . Wnt signaling is also implicated in posterior patterning and germband elongation [2-4], but despite its expression in the posterior growth zone, Wnt1/wg alone is not responsible for these functions [1-3]. Tribolium contains additional Wnt family genes that are also expressed in the growth zone . After depleting Tc-WntD/8 we found a small percentage of embryos lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced the penetrance of this phenotype. Seeking alternative methods to deplete Wnt signal, we performed RNAi with other components of the Wnt pathway including wntless (wls), porcupine (porc), and pangolin (pan). Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1 RNAi, but not Tc-WntD/8 RNAi, indicating that Tc-WntD/8 function is Tc-wls independent. Depletion of Tc-porc and Tc-pan produced embryos resembling double Tc-Wnt1,Tc-WntD/8 RNAi embryos, suggesting that Tc-porc is essential for the function of both ligands, which signal through the canonical pathway. This is the first evidence of functional redundancy between Wnt ligands in posterior patterning in short-germ insects. This Wnt function appears to be conserved in other arthropods  and vertebrates [7-9].
Project description:During the anterior-posterior fate specification of insects, anterior fates arise in a nonelongating tissue (called the "blastoderm"), and posterior fates arise in an elongating tissue (called the "germband"). However, insects differ widely in the extent to which anterior-posterior fates are specified in the blastoderm versus the germband. Here we present a model in which patterning in both the blastoderm and germband of the beetle <i>Tribolium castaneum</i> is based on the same flexible mechanism: a gradient that modulates the speed of a genetic cascade of gap genes, resulting in the induction of sequential kinematic waves of gap gene expression. The mechanism is flexible and capable of patterning both elongating and nonelongating tissues, and hence converting blastodermal to germband fates and vice versa. Using RNAi perturbations, we found that blastodermal fates could be shifted to the germband, and germband fates could be generated in a blastoderm-like morphology. We also suggest a molecular mechanism underlying our model, in which gradient levels regulate the switch between two enhancers: One enhancer is responsible for sequential gene activation, and the other is responsible for freezing temporal rhythms into spatial patterns. This model is consistent with findings in <i>Drosophila melanogaster</i>, where gap genes were found to be regulated by two nonredundant "shadow" enhancers.